The liver represents the body's central organ for amino acid and carbohydrate metabolism. As a pathway which bridges the metabolism of these two nutrient classes, hepatic gluconeogenesis plays an important role in homeostasis in both the normal and diabetic patient. Diabetes mellitus is a disease characterized by aberrations in amino acid and carbohydrate metabolism, including severe hyperglycemia. The excess plasma glucose has primarily two possible origins, decrease degradation due to the lack of insulin and increased synthesis due to stimulation of gluconeogenesis. The experiments described in the first half of this proposal will determine if hepatic amino acid transport is an important regulatory step in the conversion of plasma amino acids to plasma glucose. These studies will utilize the in vitro model system of rat hepatocytes, isolated by collagenase perfusion of the liver and maintained in primary culture for up to 24 h. The cells will be used to measure the rate of amino acid-dependent gluconeogenesis and the rate of transport into hepatocytes for each amino acid. These experiments should provide new insight into the role of hepatic amino acid transport in the development of diabetic-hyperglycemia. In addition to these metabolic studies, experiments outlined in the second half of the proposal are designed to investigate the mechanisms by which glucagon and diabetes cause a stimulation of amino acid transport via System A. This increase in activity is known to occur by processes both dependent and independent on denovo protein synthesis. Membrane protein phosphorylation will be studied as a possible mechanism for rapid activation of transport in the absence of protein synthesis. The protein synthesis-dependent portion of glucagon's action on System A will be investigated with the aid of macromoculecular synthesis inhibitors such as cycloheximide, actinomycin, and tunicamycin. Also, both affinity and photoaffinity amino acid derivatives will be used to test for irreversible inhibition of System A transport. The long-term goal of these latter experiments is to identify the System A binding protein.